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The in vitro generation of lung and airway progenitor cells from human pluripotent stem cells

Nature Protocols volume 10, pages 413–425 (2015)Cite this article

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Abstract

Lung and airway epithelial cells generated in vitro from human pluripotent stem cells (hPSCs) have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human lung development. Here we describe a strategy for directed differentiation of hPSCs into developmental lung progenitors, and their subsequent differentiation into predominantly distal lung epithelial cells. The protocol entails four stages that recapitulate lung development, and it takes ∼50 d. First, definitive endoderm (DE) is induced in the presence of high concentrations of activin A. Subsequently, lung-biased anterior foregut endoderm (AFE) is specified by sequential inhibition of bone morphogenetic protein (BMP), transforming growth factor-β (TGF-β) and Wnt signaling. AFE is then ventralized by applying Wnt, BMP, fibroblast growth factor (FGF) and retinoic acid (RA) signaling to obtain lung and airway progenitors. Finally, these are further differentiated into more mature epithelial cells types using Wnt, FGF, cAMP and glucocorticoid agonism. This protocol is conducted in defined conditions, it does not involve genetic manipulation of the cells and it results in cultures in which the majority of the cells express markers of various lung and airway epithelial cells, with a predominance of cells identifiable as functional type II alveolar epithelial cells.

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Figure 1: Schematic illustration of the protocol for lung and airway progenitor cell generation.
Figure 2: Culture morphology between days −1 and 15.
Figure 3: Variability in hPSC differentiation potential associated with DE dissociation timing.
Figure 4: Expression of FOXA2 NKX2.1 SOX2 in RUES2 cells at days 15 and 25 of differentiation.
Figure 5: Differentiation of hPSC-derived lung and airway progenitors at ∼d50.
Figure 6: Expression of NKX2.1, FOXA2 and SP-B at ∼d50 in RUES2, SVhiPS1 and SVhiPS2 cells.
Figure 7: Dynamics of ∼d50 and 3-month cultures of differentiated RUES2 cells.

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Acknowledgements

This work was supported by Price Center for Comprehensive Chest Care at Columbia University Medical Center, and by a US National Institutes of Health grant 1R01HL120046 to H.-W.S.

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Authors and Affiliations

  1. Columbia Center for Translational Immunology, Columbia University Medical Center, New York, New York, USA

    Sarah X L Huang, Michael D Green, Ana Toste de Carvalho, Melanie Mumau, Ya-Wen Chen & Hans-Willem Snoeck

  2. Department of Medicine, Columbia University Medical Center, New York, New York, USA

    Sarah X L Huang, Michael D Green, Ana Toste de Carvalho, Melanie Mumau, Ya-Wen Chen & Hans-Willem Snoeck

  3. Department of Developmental and Regenerative Biology, Black Family Stem Cell Institute, Experimental Therapeutic Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Sunita L D'Souza

  4. Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York, USA

    Hans-Willem Snoeck

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  1. Sarah X L Huang
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Contributions

S.X.L.H. developed the lung and airway differentiation protocol and co-wrote the manuscript; M.D.G. developed the AFE generation protocol; A.T.de C., M.M. and Y.-W.C. contributed to the development of the protocol; S.L.D. provided cells used in differentiation assays; H.-W.S. developed the concept, contributed to protocol development and co-wrote the manuscript with S.X.L.H.

Corresponding author

Correspondence to Hans-Willem Snoeck.

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Competing interests

The authors have filed patent applications PCT/US11/33751 and IRCU13340.

Integrated supplementary information

Supplementary Figure 1 Effect of hPSC maintenance or DE dissociation method on CXCR4/EPCAM expression profile.

Differently maintained RUES2 cells (distinguished from each other by passage number) were differentiated towards DE in parallel as two biological repeats (Experiment 1 and 2). (a) When properly maintained, p18 RUES2 cells made almost pure DE at both day 4 (not shown) and day 4.75 (right plot) of differentiation. DE was dissociated by gently flicking the tubes for 1 min. (b) When p22 RUES2 cells were maintained with a feeder-RUES2 density ratio lower than that for RUES2 cells shown in panel a, they showed reduced capacity for DE specification at both day 4 (not shown) and day 4.75 (middle plot). Notably, forceful pipetting the trypsin-cell suspension for ~30s with 1000 μL pipette tips (designated“partial pipetting”) caused a 20% decrease in the percentage of CXCR4+ population at day 4.75 (right plot). (c) P22 RUES2-derived DE, from the same experimental low-attachment plate as cells shown in middle panels, was dissociated with both methods by another lab member in parallel. The results by “flicking method” (second plot) and “partial pipetting” (third plot) were repeated. In addition, the right plot of this panel shows that forceful pipetting the trypsin-cell suspension for 1-1.5 min until all the EBs were dissociated (designated “complete pipetting”) caused a >40% decrease in CXCR4+ population.

Note: 1) the results from this figure do not suggest a correlation between passage number and endoderm induction efficiency. When properly maintained, a higher passage RUES2 (i.e., p25) still made nearly pure endoderm at day 4 of differentiation (not shown). Specifically, the p18 RUES2 cells shown in the above experiment were properly maintained further to passage 22 for a differentiation. The endoderm derived from these p22 cells showed a nearly pure CXCR4, c-KIT and EpCAM at both d4 and d4.75 (data not shown). 2) The results of this experiment suggest that “poor hPSC maintenance” and “incorrect endoderm dissociation method” may produce similar CXCR4 and EpCAM expression profile by flow cytometry.

Supplementary Figure 2 Expression of FOXA2/NKX2.1/P63 at day 25 of differentiation examined by immunofluorescence.

(a) Culture protocol for cells shown in panels b-c. (b) Day 25 cells derived from RUES2 cells. (c) Day 25 cells derived from mRNA hiPS cells.

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Huang, S., Green, M., de Carvalho, A. et al. The in vitro generation of lung and airway progenitor cells from human pluripotent stem cells. Nat Protoc 10, 413–425 (2015). https://doi.org/10.1038/nprot.2015.023

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